GENETIC ANALYSIS OF THYMOCYTE DEVELOPMENT

胸腺细胞发育的遗传分析

• 批准号: 6432580
• 负责人: PAUL E LOVE
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432580
• 关键词: CD3 molecule; T cell receptor; T lymphocyte; biological signal transduction; developmental genetics; gene expression; gene targeting; genetic regulation; laboratory mouse; leukocyte activation /transformation; leukopoiesis; molecular cloning; polymerase chain reaction; protein structure function; receptor expression

Research is directed at investigating the cellular and genetic events that control T cell development. Transgenic and gene- targeting approaches are used to analyze the function of known genes and various molecular/genetic techniques (e.g.,RT-PCR, gene cloning) are used to identify novel genes that participate in thymocyte development. Current studies are directed at three major areas: (I) Role of T cell antigen receptor (TCR) signal transduction in thymocyte maturation. In mature T cells, the TCR transduces signals important for T cell activation and cell mediated immunity. In immature T cells, TCR signals are required for thymocyte development and for thymocyte(positive/negative) selection. The TCR is composed of multiple signal transducing subunits (the CD3 chains:gamma, delta and epsilon, and the zeta chain). These subunits couple the TCR to intracellular signal transduction pathways via conserved functional sequences termed Immunoreceptor Tyrosine based Activation Motifs(ITAMs)located in their cytoplasmic domains. To determine if the TCR signal transducing subunits perform distinct or analogous functions in development, we: a)generated zeta deficient and CD3-epsilon deficient mice by gene targeting, b) genetically reconstituted these mice with transgenes encoding wild-type or signaling- deficient forms of zeta and CD3-epsilon, and c) characterized the developmental and functional consequences of these alterations in TCR signaling potential. These studies revealed that expression of zeta-family and CD3 chains is necessary for normal T cell development but that no individual TCR signaling motif (ITAM) is specifically required. Thus the TCR signaling subunits appear to be functionally redundant. However, a direct relationship was observed between the number of TCR ITAMs and the strength of the TCR signal,indicating that the multiple ITAMs within the TCR function to amplify the signaling response. These results demonstrate a previously unappreciated role for the multiple TCR ITAMs, particularly during thymocyte selection, and identify an important function for signal amplification in generation of the mature T cell repertoire. (II) The role of other signal transducing proteins in T cell development has been examined by generating transgenic/knockout mice. These include CD5, a surface receptor distinct from the TCR that also contains an ITAM-like sequence but acts as a negative regulator of TCR signaling, and CD69, an early marker of T cell activation. (III) Genes that have potential functions in thymocyte development and/or T cell activation have been identified. A new lymphoid- specific kinase, Txk cloned in the lab has been shown to function in the TCR signaling pathway leading to calcium mobilization. A similar approach has been employed to identify other novel genes that may specify commitment of multipotent progenitor cells to the T cell lineage. - Immunology, T cells, Development, Transgenes, Gene trageting, Signal transduction.

研究旨在研究控制T细胞发育的细胞和遗传事件。转基因和基因靶向方法用于分析已知基因的功能，并使用各种分子/遗传技术（例如RT-PCR，基因克隆）来识别参与胸腺细胞发育的新型基因。当前的研究针对三个主要领域：（i）T细胞抗原受体（TCR）信号转导在胸腺细胞成熟中的作用。在成熟的T细胞中，TCR传递信号对于T细胞激活和细胞介导的免疫力很重要。在未成熟的T细胞中，胸腺细胞发育和胸腺细胞（正/阴性）选择需要TCR信号。 TCR由多个信号转导亚基（CD3链：伽玛，三角洲和埃普西隆和Zeta链）组成。这些亚基通过位于其细胞质结构域中的基于免疫感受器酪氨酸的活化基序（ITAMS），将TCR与细胞内信号转导通路相结合。 To determine if the TCR signal transducing subunits perform distinct or analogous functions in development, we: a)generated zeta deficient and CD3-epsilon deficient mice by gene targeting, b) genetically reconstituted these mice with transgenes encoding wild-type or signaling- deficient forms of zeta and CD3-epsilon, and c) characterized the developmental and functional consequences of these alterations in TCR信号电位。这些研究表明，对于正常的T细胞发育，Zeta-Family和CD3链的表达是必需的，但是不需要单独的TCR信号基序（ITAM）。因此，TCR信号亚基在功能上似乎是冗余的。但是，在TCR ITAM的数量和TCR信号的强度之间观察到了直接关系，这表明TCR函数中的多个ITAMS可以扩增信号响应。这些结果证明了多个TCR ITAM的先前未见作用，尤其是在胸腺细胞选择过程中，并确定了成熟T细胞库生成信号扩增的重要功能。 （ii）通过产生转基因/基因敲除小鼠，已经检查了其他信号转导蛋白在T细胞发育中的作用。其中包括与TCR不同的表面受体CD5，它也包含类似ITAM的序列，但充当TCR信号的负调节剂，而CD69是T细胞激活的早期标记。 （iii）已经鉴定出在胸腺细胞发育和/或T细胞激活中具有潜在功能的基因。已显示在实验室中克隆的一种新的淋巴特异性激酶TXK在TCR信号传导途径中起作用，导致钙动员。已经采用了一种类似的方法来识别其他新型基因，该基因可能指定多能祖细胞对T细胞谱系的承诺。 - 免疫学，T细胞，发育，转基因，基因trageting，信号转导。